This proposal investigates the interactions of human papillomavirus type 16 (HPV16) oncoproteins and the transforming growth factor-beta (TGF-beta) signaling pathway that lead to TGF-beta resistance of HPV16-immortalized human keratinocytes (HKc/HPV16) at late stages of premalignant progression in vitro. In addition, the molecular basis for the marked inhibition of HPV16 P97 promoter activity by TGF-beta will be investigated. Our preliminary results and those of others show that the HPV16 oncoproteins E6 and E7 and the TGF-beta signaling pathway exist in a balance, in which they exert opposite effects on each other and on cell proliferation: TGF-beta inhibits HPV16 early gene transcription, lowering the levels of E6/E7 in cells immortalized by full-length HPV16 DNA. On the other hand, E6/E7 inhibit TGF-beta signaling, inducing resistance to the growth inhibitory properties of TGF-beta. Our specific aims are designed to investigate the details of these interactions in our in vitro model for HPV16-mediated multistep carcinogenesis of HKc. Progression of HKc/HPV16 toward a malignant phenotype in culture is constantly associated with the acquisition of TGF-beta resistance culminating, at late stages, with a loss of expression of the TGF-beta receptor type I. Acute expression of HPV1 6 E6/E7 from retroviral vectors produces an increase in Smad3 levels. Smad3 levels are even higher in established HKc/HPV16 lines, and increase further during progression. Stabilization and inactivation of Smad3, or binding and inactivation of phospho-Smad3 are two of the possible mechanisms by which E7 and perhaps also E6 may inhibit TGF-beta signaling. We propose to investigate in detail the mechanisms by which HPV16 E6/E7 inhibit TGF-beta activities, beginning with a mutagenesis analysis of E6 and E7, to determine the regions/activities of E6/E7 needed for this effect. We will also investigate the basis for the loss of TGF-beta receptor type I at late stages of in vitro progression of HKc/HPV16. In addition, we have defined the TGF-beta-responsive segment of the HPV16 upstream regulatory region, determined that NF-l binding sites mediate most of TGF-beta inhibition, and formulated a plan to further investigate the inhibition of this promoter by TGF-beta.